Conjugated secondary antibody

Paraffin sections of quadriceps muscle tissue were used to detect the subcellular localization of SRF by immunofluorescence staining as described previously,8 (link) with the antibody against SRF and corresponding conjugated secondary antibody (Sigma). The nuclei were indicated by 4′,6-diamidino-2-phenylindole staining. Images were captured under a Leica DMI 4000 B fluorescence microscope.

The Bcl2 protein expression level in kidney tissue from the control and experimental mice groups were detected following the standard practice of immunofluorescence using an anti-Bcl2 primary antibody (Santa Cruz Biotechnology, Inc., USA), FITC (Fluorescein-5-isothiocyanate) conjugated secondary antibody (Sigma, USA), and counterstained using DAPI (4′,6-diamidino-2-phenylindole). The images were photographed using an inverted fluorescence microscope (Carl Zeiss Axio Vert A1-FL-LED) at 10× magnification. The intensity of fluorescence was also measured using Image J software under a specific plugin size [23 (link)].

Immunoblot analysis was conducted by using anti-cytochrome c, anti-bax, anti-bcl-2, anti-p53 and anti-top-II antibodies (Santa Cruz Biotechnology, USA) and anti-cleaved caspases-3 antibodies (BD Bioscience, USA). Alkaline phosphatase was used as conjugated secondary antibody (Sigma, USA) [35] (link). Band intensities were analyzed using Image J software. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) served as the house-keeping gene, the expression of which was studied with the aid of anti-GAPDH monoclonal antibody (Santa Cruz Biotechnology, USA).